Intracellular O 2. Respiratory Burst Imaging Kit. Item No Customer Service * Technical Support

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1 Intracellular O 2 Respiratory Burst Imaging Kit Item No Customer Service * Technical Support

2 TABLE OF CONTENTS GENERAL INFORMATION 3 Materials Supplied 4 Precautions 4 If You Have Problems 4 Storage and Stability 4 Materials Needed but Not Supplied INTRODUCTION 5 Background 6 About This Assay PRE-ASSAY PREPARATION 7 Reagent Preparation ASSAY PROTOCOL 8 Plate Set Up 9 Performing the Assay PERFORMANCE CHARACTERISTICS 10 Representative Staining Results Materials Supplied GENERAL INFORMATION Kit will arrive packaged as a 4 C kit. For best results, remove components and store as stated below. Item Number Item 100 Tests Quantity/Size MitoImage TM NanO 2 Assay Reagent 1 vial/lyophilized powder 4 C Storage Cell-Based Assay Buffer Tablet 1 vial/1 tablet Room Temperature Cell-Based Assay Hoechst Dye 1 vial/50 µl -20 C Cell-Based Assay Glucose Oxidase 1 vial/2 mg -20 C If any of the items listed above are damaged or missing, please contact our Customer Service department at (800) or (734) We cannot accept any returns without prior authorization. RESOURCES 11 Troubleshooting 12 References 13 Related Products! WARNING: This product is for laboratory research use only: not for administration to humans. Not for human or veterinary diagnostic or therapeutic use. 14 Warranty and Limitation of Remedy 15 Plate Template 16 Notes GENERAL INFORMATION 3

3 Precautions Please read these instructions carefully before beginning this assay. For research use only. Not for human or diagnostic use. If You Have Problems Technical Service Contact Information Phone: (USA and Canada only) or Fax: Hours: M-F 8:00 AM to 5:30 PM EST In order for our staff to assist you quickly and efficiently, please be ready to supply the lot number of the kit (found on the outside of the box). Storage and Stability This kit will perform as specified if stored as directed in the Materials Supplied section on page 3 and used before the expiration date indicated on the outside of the box. When handling the solution or stained samples, it is recommended that they be shielded from light. Background INTRODUCTION Generation of a respiratory (or oxidative) burst by neutrophils and macrophages is an essential component of the innate immune response. Central to the respiratory burst is the conversion of molecular oxygen (O 2 ) to reactive oxygen and nitrogen species, which are involved in the killing of pathogens. In this context, O 2 is consumed foremost by NADPH oxidase, which produces superoxide. Coincident with infection, additional intracellular oxygen may be utilized by numerous other enzymes, including nitric oxide synthase, lipoxygenases, and cyclooxygenases. The Intracellular O 2 Respiratory Burst Imaging Kit facilitates the visualization of intracellular oxygen depletion resulting from this critical process. As well as investigating agents that induce and modulate this process, the kit can also be used to study the various immune-related disorders associated with dysregulated NADPH oxidase activity. 1,2 These measurements are enabled by MitoImage NanO 2, a phosphorescent probe developed by Luxcel Biosciences to monitor O 2 levels inside live eukaryotic cells by fluorescence imaging. 3 The probe is cell permeable and can be taken up and retained by cells. Once inside cells, probe phosphorescence is quenched by O 2 in a non-chemical, reversible manner; thus, the fluorescent signal is inversely proportional to intracellular O 2 levels. The use of MitoImage NanO 2 provides a fast method of monitoring oxygen depletion without the technical challenges and throughput limitations associated with micro-needles. Materials Needed But Not Supplied 1. A 96-well plate for culturing cells. 2. A microscope equipped with a UV filter set to visualize Hoechst staining of nuclei at excitation and emission at 350 nm and 461 nm, respectively. For the MitoImage NanO 2 signal, optimal excitation is nm and emission is nm. A filter which excites at 505 nm or 540 nm can be used but it is less efficient. 3. Distilled water. 4 GENERAL INFORMATION INTRODUCTION 5

4 About This Assay Cayman s Intracellular O 2 Respiratory Burst Imaging Kit employs MitoImage NanO 2 for detecting O 2 in living cells, as well as Hoechst Dye for counterstaining of nuclei. The assay is performed in an unsealed environment where the back diffusion of ambient oxygen is not restricted, and in this way differs from Cayman s Oxygen Consumption Rate Assay Kit (MitoXpress -Xtra HS Method) (Item No ), which is performed under a sealed environment in which the exchange of O 2 is limited. This kit is intended to be used for detecting oxygen depletion resulting from an induced respiratory burst. Reagent Preparation PRE-ASSAY PREPARATION 1. Preparing the MitoImage NanO 2 Staining Solution Reconstitute the contents in the vial (Item No ) with 100 µl of sterile distilled water. Mix well. Prepare a Staining Solution by diluting the Stock Solution 1:100 in the culture medium you are using for your cells. For example, if you are making 10 ml of MitoImage NanO 2 Staining Solution, add 100 µl of the MitoImage NanO 2 Stock Solution to 10 ml of culture medium. Sufficient MitoImage NanO 2 is provided to make up to 10 mls of Staining Solution. The MitoImage NanO 2 Staining Solution should be prepared just before use. The reconstituted Staining Solution is stable for up to four weeks if stored at 4 C. 2. Assay Buffer Preparation Dissolve one Cell-Based Assay Buffer Tablet (Item No ) in 100 ml of distilled water. This buffer should be stable for approximately one year at room temperature. 3. Cell-Based Assay Hoechst Dye Staining Solution Preparation To make a Hoechst Dye staining solution, add 2 µl of Hoechst Dye to 1 ml of the culture medium used in your experiment. Mix well. The Staining Solution should be prepared just before use. 4. Glucose Oxidase Stock Solution Preparation The Cell-Based Assay Glucose Oxidase is included in the kit to be used as an optional positive control. Addition of the reagent at the recommended concentration, in the presence of glucose, will give a rapid increase in probe signal. Prior to use, reconstitute the contents in the vial (Item No ) with 0.2 ml of distilled water. The reconstituted stock solution will be stable for two months when stored at -20 C. 6 INTRODUCTION PRE-ASSAY PREPARATION 7

5 Plate Set Up ASSAY PROTOCOL There is no specific pattern for using the wells on the plate. A typical experimental plate will include wells without cells, wells with cells treated with experimental compounds, and wells of untreated cells. We recommend that each treatment be performed in triplicate and that you record the contents of each well on the template sheet provided (see page 15). Performing the Assay 1. Seed cells in a 96-well plate at a density of cells/well in 100 µl of culture medium. Culture the cells in a CO 2 incubator at 37 C for 24 hours. 2. Aspirate the supernatant. Add 100 µl of MitoImage TM NanO 2 Staining Solution to each well and incubate the cells for three hours to overnight. NOTE: For cells in suspension, centrifuge the plate at 1,000 rpm for five minutes before aspirating the supernatant. 3. Aspirate the supernatant and wash the cells two times with the culture medium. NOTE: For cells in suspension, centrifuge the plate at 1,000 rpm, for five minutes before aspirating the supernatant. 4. Add 100 µl of culture medium with or without test compounds (each sample should be run in duplicate or triplicate) and incubate the cells in a CO 2 incubator at 37 C for hours, or for a period of time according to your experimental protocol. If you are using the included Cell-Based Assay Glucose Oxidase as a positive control, have two additional wells of cells treated with vehicle and assign them as positive control wells. 5. At the end of the experiment, centrifuge the plate for five minutes at 1,000 rpm. Aspirate the supernatant. 6. Add 100 µl of Hoechst Dye Staining Solution prepared above to each well and incubate samples at 37 C for two to five minutes. 7. Centrifuge the plate for five minutes at 1,000 rpm. Aspirate the supernatant. 8. Add 90 µl of culture medium and 10 µl of Glucose Oxidase Stock Solution to both positive control wells. Alternatively, add 90 µl of the Assay Buffer containing 100 mm glucose and 10 µl of Glucose Oxidase Stock Solution to both positive control wells. 9. Add 100 µl of Assay Buffer to each well except the postive control wells. Visualize the cells under a microscope capable of detecting fluorescence at an excitation wavelength of nm and an emission wavelength of nm. A filter which excites at 505 or 540 nm can be used but it is less efficient. Hoechst Dye staining of cell nuclei can be visualized with a UV filter set. 8 ASSAY PROTOCOL ASSAY PROTOCOL 9

6 PERFORMANCE CHARACTERISTICS NOTE: This kit uses MitoImage NanO 2 to detect changes in intracellular oxygen levels in response to respiratory burst. For analysis of changes in oxygen consumption or respiration rate, we recommend using Cayman s Oxygen Consumption Rate Assay Kit (MitoXpress - Xtra HS Method, Item No ). Representative Staining Results Troubleshooting RESOURCES Problem Possible Causes Recommended Solutions High fluorescence signal in all wells including vehicle controls A. Overgrowth of cells B. Culture medium containing phenol red not completely removed A. Use lower cell density B. Wash the cells with the Assay Buffer Low or no fluorescence signal Cells are not healthy Use healthy cells A B Decreased fluorescence in control well Photobleaching of probe A. Reducing lamp intensity B. Reducing illustration time/ frequency C D Figure 1. LPS significantly decreases intracellular O 2 levels in RAW cells, indicated by an increase in NanO 2 signal. RAW264.7 cells were seeded in a 96-well plate at a density of 20,000 cells/well in 100 µl culture medium and incubated in a cell culture incubator overnight. The next day, cells were labeled with MitoImage TM NanO 2, as described in the booklet. Cells were then cultured in 100 µl culture medium containing either vehicle (upper panels) or 1 µg/ml LPS (lower panels) for 48 hours. Left panels show staining of nuclei by Hoechst Dye. Panels on the right show fluorescence signal from MitoImage TM NanO 2 (signal inversely proportional to O 2 concentration) in fields corresponding to those in left panels. 10 PERFORMANCE CHARACTERISTICS RESOURCES 11

7 References 1. Dinauer, M.C. and Orkin, S.H. Chronic granulomatous disease. Molecular genetics. Hematol. Oncol. Clin. North. Am. 2(2), (1988). 2. Moraes, T.J., Zurawska, J.H., and Downey, G.P. Neutrophil granule contents in the pathogenesis of lung injury. Curr. Opin. Hematol. 13(1), (2006). 3. Dmitriev, R.I., Zhdanov, A.V., Nolan, Y.M., et al. Imaging of neurosphere oxygenation with phosphorescent probes. Biomaterials 34(37), (2013). Related Products Hydrogen Peroxide Cell-Based Assay Kit - Item No Nitric Oxide Cell-Based Assay Kit - Item No Oxygen Consumption/Glycolysis Dual Assay Kit - Item No Oxygen Consumption/MitoMembrane Potential Dual Assay Kit - Item No Oxygen Consumption Rate Assay Kit (MitoXpress -Xtra HS Method) - Item No RESOURCES RESOURCES 13

8 Warranty and Limitation of Remedy Cayman Chemical Company makes no warranty or guarantee of any kind, whether written or oral, expressed or implied, including without limitation, any warranty of fitness for a particular purpose, suitability and merchantability, which extends beyond the description of the chemicals hereof. Cayman warrants only to the original customer that the material will meet our specifications at the time of delivery. Cayman will carry out its delivery obligations with due care and skill. Thus, in no event will Cayman have any obligation or liability, whether in tort (including negligence) or in contract, for any direct, indirect, incidental or consequential damages, even if Cayman is informed about their possible existence. This limitation of liability does not apply in the case of intentional acts or negligence of Cayman, its directors or its employees. Buyer s exclusive remedy and Cayman s sole liability hereunder shall be limited to a refund of the purchase price, or at Cayman s option, the replacement, at no cost to Buyer, of all material that does not meet our specifications. Said refund or replacement is conditioned on Buyer giving written notice to Cayman within thirty (30) days after arrival of the material at its destination. Failure of Buyer to give said notice within thirty (30) days shall constitute a waiver by Buyer of all claims hereunder with respect to said material. For further details, please refer to our Warranty and Limitation of Remedy located on our website and in our catalog A B C D E F G H 14 RESOURCES RESOURCES 15

9 NOTES This document is copyrighted. All rights are reserved. This document may not, in whole or part, be copied, photocopied, reproduced, translated, or reduced to any electronic medium or machine-readable form without prior consent, in writing, from Cayman Chemical Company. 08/07/2014, Cayman Chemical Company, Ann Arbor, MI, All rights reserved. Printed in U.S.A. 16 RESOURCES

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